How much do field mice prefer dwarf bamboo seeds? Two‐choice experiments between seeds of Sasa borealis and several tree species on the forest floor

Abstract Bambusoideae is a taxon of mass‐flowering monocarpic perennials with a long life cycle. Forest ecosystems are affected by Bambusoideae seeding and death events in various ways, including an increased abundance of Apodemus spp. The utilization and preference of dwarf bamboo seeds over tree seeds by field mice remain elusive. Therefore, we aimed to determine whether field mice prefer dwarf bamboo to tree seeds. We examined one dwarf bamboo species (Sasa borealis) against four tree species with varying acorn/fruit traits (Castanea crenata, Quercus crispula, Fagus crenata, and Lindera triloba). The seeds were placed in a container in a forest among dead S. borealis culms, with an automatic camera monitoring the setup. The examined seeds were mainly foraged by two field mouse species, Apodemus speciosus and Apodemus argenteus, with preference in the following order: C. crenata, L. triloba, S. borealis, F. crenata, and Q. crispula. Our findings indicated that during S. borealis mast seeding years, predation pressure on F. crenata and Q. crispula seeds could be considerably reduced. This suggests that mast seeding might disrupt the normal pattern of survival, and seed dispersal patterns, potentially altering the forest vegetation composition.


| INTRODUC TI ON
Bambusoideae, including bamboo and dwarf bamboo, exhibit largescale flowering, seeding, and death (hereinafter referred to as "mast seeding"), in a cycle of three to over 120 years (Janzen, 1976).For example, the dwarf bamboo species Sasa borealis (Hack.)Makino et Shibata undergoes a flowering cycle every 120 years.This flowering is often reported to be synchronized intraspecifically (e.g., Cho et al., 2017;Nagata & Tamura, 2014;Niiyama et al., 2021).Such synchronization factor is evolutionarily grounded in the principle of predator satiation, where producing many seeds simultaneously increases the probability of escaping predators (Janzen, 1971).S. borealis generally germinates 2 years after seeding, marking the initiation of the subsequent generation (Nakagawa et al., 2023).
The dwarf bamboo, a type of Bambusoideae that covers the forest floor, provides a habitat for field mice.These mice prefer dense forest floors for foraging and as an escape zone to protect themselves from predators (Sakamoto et al., 2012;Teixeira et al., 2017;Wada, 1993).Its evergreen leaves are a major food resource for sika deer (Cervus nippon [Temminck, 1838]), particularly during winter (Koizumi et al., 2006;Takatsuki, 1983;Tanaka et al., 2008).Dwarf bamboo is also considered a strong inhibitor of forest regeneration due to its sunlight-blocking dense and long-term growth (Doležal et al., 2009;Hasegawa & Fujibe, 2015;Nakashizuka & Numata, 1982;Uno et al., 2019).These characteristics suggest that bamboo is an important component of forest ecology, implying that the impact of a bamboo mast seeding event would be significant.
One of the impacts of a bamboo mast seeding event is a large seed supply on the forest floor.Bamboo seeds are considered highly valuable because their crystalline morphology and starch structure are comparable to rice, although the content is slightly lower (Ai et al., 2016).Historically, bamboo seeds were used as food by humans (Kiruba et al., 2007).Birds and rodents have been reported consuming bamboo seeds in the wild (Areta et al., 2009;Areta & Cockle, 2012;Franklin, 2005;Kitzberger et al., 2007;Lebbin, 2006).In particular, the mast seeding of Bambusoideae leads to a rapid outbreak of rodent populations (Bovendorp et al., 2020;González et al., 2000;Ito, 1975;Suzuki et al., 2022).
Nevertheless, studies on rodents' preferences for bamboo seeds over other seeds are few, with only one reported from Argentina by Kitzberger et al. (2007).They conducted experiments with seeds of higher and lower food quality than bamboo seeds and found that the presence of bamboo seeds altered the predation pressure on seeds of varying qualities.Forest rodents are not only seed predators but also seed dispersers owing to their seed-caching habits (Klinger & Rejmanek, 2009).Therefore, understanding how the abundant bamboo seed supplied by mast seeding alters the food selection of rodents and its potential effects on other plants in the forest ecosystem is essential.
Consequently, in this study, we conducted field-feeding tests using seeds of S. borealis (Figure 1c), a dwarf bamboo species, and four other tree species (Figure 1d 5 years after mast seeding.In forest areas where the forest floor is covered by dwarf bamboo, tree regeneration is hindered due to its direct impact.Therefore, after the disappearance of dwarf bamboo by mast seeding, tree renewal is expected to proceed. To clarify this cycle, the apparent competition between the seeds must be determined.In a previous study (Suzuki & Kajimura, 2023) conducted at the same site, only S. borealis seeds were tested in the forest to identify the foraging species.The results showed that field mice, the large Japanese field mouse Apodemus speciosus (Temminck, 1844) (Figure 1a) and the small Japanese field mouse A. argenteus (Temminck, 1844) (Figure 1b) forage using predation and removal (including caching).Based on these findings, this study aimed to determine the preference of seed predators for S. borealis seeds over tree seeds based on foraging rate and order.
The impact of the mast seeding of S. borealis on the forest is also discussed.

| Survey site
The study was conducted in the Takatokke district of Nagoya University Forest (in the Inabu Field affiliated with the Graduate

| Feeding test
Chestnuts of C. crenata (Figure 1d), acorns of Q. crispula (Figure 1e), beechnuts of F. crenata (Figure 1f), and seeds of S. borealis and L. triloba (Figure 1g) were used for the feeding tests.All seeds, including chestnuts, acorns, and beechnuts, are hereafter referred to as "seeds".The seeds of these four tree species selected for this study were obtained from the surrounding forest area.They exhibited evidence of rodent foraging and were readily available.S. borealis seeds were harvested in 2017 in Dando National Forest, Aichi Prefecture, threshed, and stored at room temperature (20-25°C) in our laboratory until required for further testing.The seeds of C. crenata, Q. crispula, F. crenata, and L. triloba were collected around the plot in 2022.Weight and detailed information of each seed are provided in Table 1.Seeds used in the tests were selected from sound seeds free from insect damage.Preferences among seeds were made by simultaneously comparing each tree seed with S. borealis seeds.For the tests of C. crenata or Q. crispula, a set comprised 5 g of S. borealis seeds and four seeds of C. crenata or Q. crispula.For the tests of F. crenata or L. triloba, a set comprised 2.5 g of S. borealis seeds and two seeds of F. crenata or L. triloba.These ratios of masses and numbers were based on our evidence and observation of a previous study that exclusively used S. borealis (Suzuki & Kajimura, 2023).We determined the amount of S. borealis required for approximate seed consumption in one night and the number of tree seeds available on the forest floor.Each tree seed was weighed individually before the test.
At each of the four test points (Plot-BF-IN, Plot-BF-OUT, Plot-CF-IN, and Plot-CF-OUT), a food station was established by positioning a shallow metal container (22 cm in diameter) on the ground (Figure 2).Seed sets were then placed at these food stations at each test point.The tree seeds to be tested were identified by weight and placed individually at the same test point.The following day, all the remaining seeds were collected.
The remaining S. borealis seeds were weighed.The weight of seeds utilized by the visiting animals was calculated by subtracting the remaining weight from the initial weight (5 or 2.5 g).Additionally, the remaining tree seeds were counted.The seed foraging rate was defined as the percentage of lost seeds (or weight) to initial seeds (or weight).A series of feeding tests were conducted from September 15 to November 16, 2022.To align with the timing of tree seed drop, C. crenata and Q. crispula tests were performed from September to early October (i.e., early fall), whereas F. crenata and L. triloba tests were performed from late October to November (i.e., late fall).Five tests were repeated for each tree seed (four species) at each point (four locations).
A trail camera (HYKE HCSP2; Hyke Inc., Hokkaido, Japan) was placed approximately 50 cm away from each container to monitor its contents and surroundings.The camera was set to high sensitivity to record a video after the sensor response and to capture an hourly time-lapse photograph.The videos were analyzed to identify the species of visiting animals and their seed utilization behaviors (predation or removal) at each food station.However, the videos did not permit the distinct identification of individual animals.The foraging rate and order were used to determine seed preference.
In addition, a preference for seed size within the same tree species was analyzed.No permissions were required prior to conducting this research, as only automatic recording of naturally visiting animals was used.

| Statistical analyses
The differences in the foraging rate after one night between S. borealis and each tree species were compared using the Mann-Whitney U-test.
To analyze the effects on the foraging rate of S. borealis, a generalized linear model was used, including season (early fall or late fall), plot (Plot-BF or Plot-CF), test point (IN or OUT), and tree species tested at the same time as explanatory variables.The preference of field mice in terms of foraging order was analyzed in two ways: whether S. borealis or tree seeds were foraged first in each test and the percentage of mice that foraged on S. borealis or tree seeds until all seeds of either type were gone.The latter was analyzed separately for A. speciosus and A. argenteus during the tree seed tests, and differences between the two were compared using Fisher's exact test.Comparisons of size (and weight) with tree seed foraging order were conducted using Student's t-test or Tukey's honestly significant difference test.All analysis programs were run in R v4.1.2(R Core Team, 2021).

| Visiting animals and their behaviors
In 80 tests, five different mammal species visited the food containers.

| Foraging rate of seed species
The foraging rates of each seed species after one night of testing are shown in Figure 3.For F. crenata, some sterile seeds that could not be determined from the outside view were included.This is because F. crenata nuts grow regardless of whether the embryo matures or not (Nakashizuka, 2009).Therefore, the remaining seeds TA B L E 1 Traits of seeds used in the study.The generalized linear model analysis revealed that the plot and testing with C. crenata were significantly related to the foraging rate of S. borealis (Table 2).Plot-CF was found to have a higher foraging rate than Plot-BF, and the foraging rate was also higher when tested with C. crenata at the same time.Microenvironmental differences, such as foraging outside or inside of dead culms, did not influence the foraging rates.

| Foraging order
Based on the camera images of S. borealis and tree seeds, the first to forage was C. crenata in 20 of 20 trials (100%), L. triloba in 11 of 18 trials (71%), F. crenata in 2 of 12 trials (16%), and Q. crispula in 2 of 20 trials (10%).These results excluded tests in which there was a camera malfunction.In addition, Figure 4 shows whether the two species of field mice were selected for S. borealis seeds or tree seeds until either seed was gone or until the next morning.The ratio represents the percentage of times each seed was selected out of the total number of foraging behaviors during all the trials.For A. speciosus, the foraging of C. crenata was higher (71%) than that of  (Kaneko, 2005).Then, foraging for S. borealis seeds was the same as that reported previously (Suzuki & Kajimura, 2023).These results support the hypothesis that the increase in their population after the mast seeding of S. borealis was due to increased food resources (Suzuki et al., 2022).In addition, foraging of S. borealis seeds by E. smithii, another rodent species, and N. procyonoides of the family Canidae, was newly identified.According to the observed behavior, E. smithii consumes the green part of plants and the starch of seeds (Kaneko, 2005).N. procyonoides is omnivorous and highly adaptable; it feeds on cereal, including Triticeae (Elmeros et al., 2018).Similarly, N. procyonoides was newly found to feed upon S. borealis of Poaceae on some occasions.The observation that neither of these two species was seen foraging in a previous study (Suzuki & Kajimura, 2023) may be attributed to a low encounter rate.M. itatsi was seen sniffing the container, possibly lured by rodents, its primary food source (Kaneko et al., 2013).Camel crickets forage S. borealis seeds when fed in rearing (H.Suzuki, H. Kajimura, unpublished data), and we consider the movements recorded on camera as part of a foraging behavior sequence.
Regarding the caching behavior exhibited by rodents, particularly A. speciosus and A. argenteus, both predation and removal behaviors were observed for S. borealis, while all tree seeds except for one C. crenata were removed.Rodent behavior can vary depending on whether they remove or predate on prey seeds, which is often influenced by the seed size (Vander Wall, 2003;Wang et al., 2012).In this experiment, S. borealis seeds averaging 0.0268 g were consumed in situ.However, F. crenata seeds averaging 0.1270 g or more, were removed, indicating their division for dietary use, as concluded in other seed studies (Vander Wall, 2003;Wang et al., 2012).
The results of the generalized linear model analysis for the foraging rate of S. borealis suggested that the factors affecting the foraging rate of S. borealis were the plot and the presence or absence of C. crenata.The higher foraging rate of S. borealis in Plot-CF aligns with that reported previously by Suzuki and Kajimura (2023), suggesting that Plot-CF may have fewer alternative food sources due to the specific characteristics of the forest area.The same plots were used for all tests; thus, the plot does not influence the preference intensity between the seeds.In contrast, the presence of simultaneously tested seeds, we consider that S. borealis was selected with the same preference throughout the experiment for the following discussion.

| Seed preference factors of field mice
Regarding the seed preference of field mice, the overnight foraging rate results (Figure 3) suggested that C. crenata may have the highest preference, followed by L. triloba, S. borealis, and F. crenata at similar levels, and Q. crispula at the lowest level.Preference was then judged in terms of the foraging order.The results of the first seeds foraged by mice showed that C. crenata and L. triloba had a higher foraging percentage before S. borealis, indicating a stronger preference.
Q. crispula and F. crenata were considered less preferential compared with S. borealis, as the percentage of S. borealis foraging was initially higher than that of Q. crispula and F. crenata.In addition, the percentage of rodents foraging on S. borealis seeds versus tree seeds by the time the tree seeds were fully foraged or the next morning (Figure 4)  (Hoshizaki, 2009;MEXT, 2015;Shimada et al., 2019).The energy of S. borealis is 14.9 kJ/g (Shimada et al., 2019).Compared with C. crenata (6.9 kJ/g), Q. crispula (14.3 kJ/g), and F. crenata (22.1 kJ/g), the energy content of S. borealis is not as high as that of F. crenata but is sufficient (Hoshizaki, 2009;MEXT, 2015).High carbohydrates and energy content are also observed in seeds of other bamboo species (Kumawat et al., 2014).Since S. borealis seeds are smaller than tree seeds, foragers require a larger number to ingest, but its large supply also makes it a more valuable food source.
C. crenata, which was the most preferred species, has been reported as a food source and cache target for several animals, particularly Apodemus spp.(Seiwa et al., 2002;Tanikawa, 2009).C. crenata has a lower energy content obtained per gram than S. borealis, but its larger seed size makes it preferred for caching and may result in a greater benefit.
Q. crispula is a large seed like C. crenata but is high in tannins and low in fat and proteins; thus, it has low value as a forage resource (Shimada & Saitoh, 2003, 2006).However, field experiments using Q. crispula seeds have revealed caching behavior by A. speciosus and A. argenteus, and Q. crispula was detected in the diet analysis of both species; thus, Q. crispula is utilized as a food source (Miura & Okitsu, 2006;Sato et al., 2018).Additionally, Saitoh et al. (2007) showed that A. speciosus population dynamics occur in response to Q. crispula abundance, whereas Hoshizaki and Miguchi (2005) noted that the relationship is unclear.Therefore, it is likely that environmental factors, such as the availability of other food sources, impact the value of Q. crispula utilization.In this experiment, the presence of another food source, i.e., S. borealis, may have more remarkably reduced the utilization value of Q. crispula.
F. crenata contains little defensive substance (Nakashizuka, 2009), is rich in fat, and is a high-quality food for mice (Hashizume, 1979;Hoshizaki, 2009;Sugawara, 1972).F. crenata is strongly associated with mice, and the population dynamics of Apodemus spp., particularly A. speciosus, have been reported to occur in response to its abundance (Hoshizaki & Miguchi, 2005;Miguchi, 1988).In this experiment, the preference for F. crenata was found to be similar to that of S. borealis in terms of foraging rate, but the preference was lower than that of S. borealis in terms of foraging order.This difference may be due to the ease of carrying and eating the seeds.Each F. crenata seed was larger than that of S. borealis, and mice could carry multiple S. borealis seeds when removing them, whereas only one F. crenata seed was carried at a time.Li et al. (2018) found a relationship between seed preference and husk peel ability through a field experiment in which several species of seeds were used.F. crenata is covered by a hard shell, whereas S. borealis has an easily peeled chaff.Therefore, S. borealis may have a higher preference because it is more efficient to forage.
The higher preference for L. triloba compared with S. borealis is difficult to determine because the seeds have not been studied sufficiently, and their characteristics are unknown.However, Lindera melissifolia of the same genus is known to have a higher ratio of saturated acid to unsaturated acid compared with other tree seeds, such as Quercus spp.(Connor et al., 2007).Mice have been reported to forage for the seeds of L. melissifolia when tested in the field (Martins et al., 2015).Therefore, it is likely that L. triloba also contains some elements favored by mice.
Regarding preference for L. triloba among field mouse species, A. argenteus selected 100% L. triloba, indicating a significantly stronger preference than A. speciosus for L. triloba.Similarly, A. speciosus clearly foraged more for Q. crispula than A. argenteus.A. speciosus has a higher tannin tolerance (Shimada et al., 2004).Sato et al. (2018) found that both Apodemus species forage for Q. crispula, but A. speciosus has a higher percentage of Q. crispula in their diet analysis, which is also in accordance with our results.
There was a significant difference in the size of C. crenata and L. triloba seeds foraged before and after S. borealis (Figure 5), clearly indicating that larger seeds are preferred.For example, Wang et al. (2012) showed that there was a preference for larger acorns among the same species in a seed-feeding experiment.In this experiment, the degree of preference was much more apparent when tested with S. borealis.

| CON CLUS IONS
This study, conducted over a brief period of approximately 3 months, coinciding with each seed supply timing, yielded the first data set identifying seed foragers and their preferences.
Our experiments were designed to compare S. borealis seeds with individual tree species for their reliable identification from video images.However, simultaneous testing of S. borealis with multiple tree species would better match field conditions (flora and its distribution), thereby determining more realistic seed preferences by mouse species.We plan to develop a better experimental system in the future.
Based on the reported results of mice preference, potential interactions between trees and S. borealis in forest ecosystems regarding seed foraging pressure were considered.S. borealis seeds in forests with C. crenata may have higher foraging rates and lower seedling establishment rates than those without.If the 120-year cycle of S. borealis seeding coincides with rich harvests of Q. crispula or F. crenata, the predation pressure may be reduced, increasing the establishment rate of these tree seedlings.This relationship is assumed to eventually lead to inhibited regeneration owing to the cover of dwarf bamboo, and future follow-up investigations related to the regeneration of the forests may be required.
The most frequent visitors were field mice of two species: A. speciosus (692 times) and A. argenteus (486 times).Smith's red-backed vole Eothenomys smithii(Thomas, 1905), raccoon dog Nyctereutes procyonoides (Gray, 1834), and Japanese weasel Mustela itatsi(Temminck,   1988)  also visited the area multiple times.S. borealis seeds were foraged by A. speciosus, A. argenteus, E. smithii (Video S1), and N. procyonoides (Video S2).However, E. smithii only preyed 13 times, and N. procyonoides preyed only 4 times.Therefore, we only considered foraging due to A. speciosus and A. argenteus.Tree seeds were exclusively foraged by A. speciosus (Video S3) and A. argenteus (Video S4).M. itatsi only sniffed the inside of the container.In addition, no birds visited the containers, although camel crickets (Rhaphidophoridae) were observed moving S. borealis on several occasions.Both predation (consumed in situ) and removal (removed to somewhere in the mouth) from the container were observed among A. speciosus and A. argenteus, whereas E. smithii was only observed removing, and N. procyonoides was only observed predating.S. borealis seeds were both consumed in situ and removed.As for tree seeds, all were removed except for one C. crenata seed that was partially consumed in situ.It was also observed that A. speciosus and A. argenteus carried several seeds at a time during the removal of S. borealis.
Result of general linear model analysis for the foraging rate of S. borealis.
S. borealis.L. triloba foraging was lower (40%) than S. borealis foraging but higher than Q. crispula and F. crenata foraging.As for A. argenteus, only one foraging instance was observed in C. crenata tests, but C. crenata was foraged, and in L. triloba tests, 100% of the foraging was for L. triloba.In contrast, in the Q. crispula and F. crenata tests, S. borealis was foraged more than 95% of the time.The L. triloba tests showed significant differences between A. speciosus and A. argenteus (Fisher's exact test, p < .05).C. crenata and L. triloba, the most foraged species, were compared by the difference in weight between seeds foraged before S. borealis, seeds foraged after S. borealis, and seeds left behind (Figure5).For both species, seeds foraged before S. borealis were significantly heavier than those foraged after S. borealis (C.crenata:4 | DISCUSS ION4.1 | Visitors and seed foragersTwo species of field mice, A. speciosus and A. argenteus, were the most abundant foragers of seeds.This result is consistent with the fact that these two species of field mice mainly forage on seeds F I G U R E 3 Violin plot representing the distribution of the foraging rate of each seed species.The white circles in the box plots indicate the mean.***: statistically significant at p < .005using the Mann-Whitney U-test with S. borealis.TA B L E 2 *Statistically significant at p < .05. ***Statistically significant at p < .001.
Percentage of observation frequency of which seed of S. borealis or trees was selected by two species of field mice until either seed was gone or the next morning, whichever occurred first.*Statistically significant at p < .05using Fisher's exact test.Lindera triloba.The white circles in the box plots indicate the mean.
C. crenata increased the foraging rate for S. borealis, suggesting that C. crenata may have been an attractant for mice.Although C. crenata should be considered for preference intensity between the F I G U R E 4 ***Statistically significant at p < .005using Student's t-test.Different letters indicate significant differences between seeds at p < .05using Tukey's honestly significant difference test.
suggests a high preference for C. crenata and L. triloba and a low preference for Q. crispula and F. crenata.The two viewpoints on for-